3D living neural networks

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Project Summary/Abstract Mental disease, including schizophrenia, depression and autism spectrum disorders, are still poorly understood, although it is clear that they mostly represent cortical disorders. The cortex is the primary site of higher mental functions, yet despite extensive research there is still no unified theory of how the cortex works. This is partly due to the fact that neuroscientists have limited tools required for precise repeatable studies of neural circuit formation and for studies of the mechanisms that control plasticity and repair. Most research completed to date relies on 2D cell cultures or studies of live brains. Some ability to control cellular distribution within 2D cultures has been demonstrated by patterning an extra-cellular protein, for example polylysine, to direct neuronal cell attachment. However, cells are subsequently washed onto the substrate. While cells generally adhere to the patterned surfaces, there is no mechanism for controlling cellular distribution with single cell resolution. Other techniques have investigated use of grids of planar electrode arrays with "cages" that only allow a single cell to be deposited in each cage. This improved the ability to unambiguously map detected signals to specific neurons, and to confine stimulation to single cells. However, this approach is not easily scalable to 3D environments, and metallic substrates do not accurately mimic a cells natural environment, which can alter cell behavior. Alternatively, studies of brain slices using photostimulation and calcium imaging circumvent many of these problems. However, this approach presents a daunting level of complexity making it challenging for neuroscientists to unravel function of the brain. Our approach offers several key benefits to neuroscience research. By taking advantage of recent advances in calcium imaging and photostimulation we remove the need for electrodes throughout our neural network to stimulate and probe connectivity. As a result a purely hydrogel scaffold can be used as the supporting structure, and as the source of channels to direct neural growth. Stereolithography enables the user to rapidly define the shape of the polymer network, step and repeat methods enable structures of arbitrary dimensions in x and y, and additive layering enables large scale axial dimensions. Furthermore, by merging stereolithography with optical trapping, micron scale control of the position of cells within the polymer structure is realized. Our commercially available optical trapping system is capable of manipulating hundreds of objects simultaneously, at high speed, and with sub-cellular resolution. When the optical trapping system is combined with stereolithography, the complete solution will allow scientists to study biological processes with unprecedented speed, resolution, and repeatability. Boulder Nonlinear Systems and the University of Colorado propose to combine their expertise in building SLMs and in SLM microscopy in a two-phase project with the ultimate goal of making dynamic 3D tissue scaffold fabrication a practical reality in neuroscience and clinical research. In the first phase we plan to build a compact, inexpensive, user-friendly inverted microscope with modules for optical trapping, and stereolithography. The device will be self-aligning and integrated with appropriate software so that it can be used, out of the box, for applications in several neurobiological projects including studies of mechanisms for plasticity and repair, drug and toxin screening, chemical and biological sensing, biocompatibility tests at the interface between a prosthetic device and human body, and research into regeneration of nerve connections for spinal cord injuries. In Phase II we will increase the throughput of the fabrication system, and extend the automation of the system. The ultimate goal is to design a tool capable of fabricating large scale neural networks and tissue scaffolds with micron resolution free of user control. Additionally in the Phase II BNS will collaborate with Olympus to design "bolt-on" optical trapping and stereolithography modules for existing Olympus microscopes. This will provide an established distribution channel for the proposed research and will allow users to utilize existing imaging modalities specialized to their individual studies.